The interferon-induced double-stranded RNA (dsRNA)-activated kinase (p68 kinase) plays a key role in the mechanism of action of interferon and is a target for inhibition by different viruses. We have cloned and characterized the human p68 and mouse p65 kinases and expressed the cDNAs in E.coli, yeast and mammalian cells. The goals of this project are to understand the mechanisms involved in the regulation of synthesis of this enzyme by interferons, its activation by dsRNA and its role in cellular metabolism and virus infection. Our cloning and characterization of the human and mouse enzymes has laid the groundwork for a detailed analysis of the structure and function of these enzymes as regulators of signal transduction, virus replication and cell growth. Accordingly, we propose experiments which should delineate structural features of the kinase required for activation by dsRNA and binding to known substrates, characterize novel substrates of the kinase, and perturb or disrupt kinase expression in cell cultures and transgenic mice. To determine how transcription of the p68 and p65 kinase genes is regulated and if interferon stimulated response elements are involved, we will characterize the 5' flanking regions of the mouse and human genes. To define the double-stranded RNA binding domain of the p68 kinase, we will use in vitro mutagenesis coupled with assays for reversion of a kinase-induced slow growth phenotype induced by the kinase in yeast. To investigate the structure of the dsRNA binding domain, we will attempt co-crystallization of this domain with dsRNA. To determine the domain of the kinase involved in substrate recognition, different mutants will be constructed using in vitro mutagenesis and analyzed by affinity chromatography and phenotype assay in yeast. To identify cellular substrates for the kinase involved in signal transduction and/or gene activation, we will use affinity chromatography assays and phenotype reversion assays in yeast. Novel cellular substrates for the kinase will be cloned using a genetic selection method in yeast to screen mammalian cDNA libraries. To determine the role of the kinase in regulating cell growth and responding to environmental stimuli, inhibitors of the kinase selected by phenotype reversion in yeast will be tested for activity in mammalian cell cultures and null mutations of the kinase established using homologous recombination in embryonic stem cells. Since the dsRNA-activated kinase regulates protein synthesis and cell growth and inhibits virus replication, this project has implications for the therapy of cancer and virus infections.